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J.D. Lai et al.
By LC-MS/MS, we detected 22 of the 25 potential N- linked Asn-X-Ser/Thr consensus sequences, and identified a total of 21 unique glycans (Table 1; for peptide coverage and glycan construction see Online Supplementary Figure S4 and Figure S5). The intensity of glyco-peptides was nor- malized to the area under the curve of non-glycosylated peptides relative to CHO-rFVIII. The predicted N-linked
glycan sites at Asn1001, Asn1005, and Asn1512 were not detected by our methods. At each occupied site, we observed significant heterogeneity in the glycan structures (Figure 5A). We next grouped glycan stuctures into either high-mannose, asialylated, partially sialylated, or fully sia- lylated. In agreement with our lectin binding data, we observed high-mannose glycans at Asn757 and Asn1300
A
BCDE
F
GHI
J
Figure 1. Immunogenic differences between BHK-rFVIII and CHO-rFVIII in HA-R593C mice. (A) Mice were immunized subcutaneously (SC) with 6 IU (0.6 μg; 240 IU/kg) of either BHK- or CHO-rFVIII biweekly for 2 weeks. Blood was collected by cardiac puncture 28 days after the first infusion. Plasma was assessed for (B) the incidence and (C) titre of FVIII-specific IgG, as well as (D) the incidence of inhibitors above 1 and 5 BU and (E) the magnitude of inhibitory activity. (F) Mice were immu- nized intravenously (IV) with 6 IU (0.6 mg; 240 IU/kg) of either BHK- or CHO-rFVIII product twice weekly for 2 weeks. The first infusion contained 1 mg of lipopolysachar- ride. Plasma samples 28 days after the first infusion were assessed for (G) the incidence and (H) titre of FVIII-specific IgG. (I) Comparison of the incidence of inhibitors above 1 BU and the (J) magnitude of inhibitory activity. Horizontal lines and error bars represent the mean and SEM, respectively. Mann-Whitney U and Fisher’s exact test were used where appropriate. *P<0.05; **P<0.01; ***P<0.001. BHK: baby hamster kidney cells; CHO: Chinese hamster ovary cells: rFVIII: recombinant factor VIII; Ig: immunoglobulin; n.s. not significant; BU: bethesda unit.
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haematologica | 2018; 103(11)